The Akt/PKB protein kinase is implicated in the control of cellcycle
progression and the suppression of apoptosis in cancercells. Here we
describe the use of a conditionally active formof Akt/PKB
(M+Akt:ER*) to study the ability of this proteinto
influence biological processes that are central to the processof
oncogenic transformation of mammalian cells. Activation of
M+Akt:ER*in Rat1 cells elicited alterations in cell
morphology and promotedanchorage-independent growth in agarose with
high efficiency.Consistent with these observations, activation of
M+Akt:ER*suppressed the apoptosis of Rat1 cells that
occurs after thedetachment of these cells from extracellular matrix.
Furthermore,activation of M+Akt:ER* was sufficient to
promote the progressionof quiescent Rat1 cells into the S and
G2-M phases of the cellcycle. In accord with this is the
observation that activationof M+Akt:ER* led to decreased
expression of the cyclin-dependentkinase inhibitor p27Kip1
with a concomitant increase in cyclin-dependentkinase-2 activity.
Perhaps surprisingly, activation of M+Akt:ER*or expression
of a constitutively active form of Akt led torapid activation of
MAP/ERK Kinase (MEK) and the extracellularsignal-regulated kinase
(ERK)/mitogen-activated protein (MAP)kinases in Rat1 cells. However,
pharmacological inhibition ofMEK by PD098059 did not inhibit the
morphological alterationsof Rat1 cells that occur after
M+Akt:ER* activation. These datasuggest that
M+Akt:ER* can activate a number of pathways inRat1 cells,
leading to significant alterations in a number ofbiological processes.
The conditional transformation systemdescribed here will allow further
elucidation of the abilityof Akt to contribute to both the normal
response of cells tomitogenic stimulation and the aberrant
proliferation observedin cancer cells.

Akt was identified, by homology to the catalytic subunit
ofthe cyclic AMP-dependent protein kinase (1)
, as a
putativeserine/threonine kinase RAC (related to cyclic
AMP-dependent
PKA3
andPKC; Ref. 2
). Because of the similarity to both PKA
and PKC,it has also been referred to as PKB(3)
. c-Akt
(4)
was independentlyidentified as the cellular homologue
of v-Akt(5)
, the oncogeneof the AKT-8
murine leukemia virus (6)
. The AKT-8 retroviruswas
originally isolated from a spontaneous thymoma in an AKRmouse and was
subsequently shown to be capable of transformingmink lung fibroblasts
in culture (7)
. After inoculation ofsusceptible mouse
strains, this virus produced thymic lymphomas(6)
, and
cells expressing v-Akt were found to be highly oncogenicwhen
injected into nude BALB/c mice (8)
. To date, three
mammalianisoforms of c-Akt have been identified (2, 5, 9, 10)
. Eachis composed of three distinct and conserved domains:
an NH2-terminalPH domain; a catalytic protein
kinase domain; and a short COOH-terminalregulatory region related to a
similar region of PKC. The Aktisoforms differ with respect to their
distribution in varioustissues as well as the levels of expression of
each isoform,although the significance of these differences remains
unclear.

c-Akt is a serine/threonine kinase (11, 12, 13)
that is
rapidlyactivated in response to a variety of cytokines and growth
factors(11, 12, 14, 15)
. The current model for the
activation ofc-Akt suggests that growth factor-mediated activation of
PI3'-kinaseleads to increased production of PI3'-lipids. Such lipids
bindto the PH domain of c-Akt, leading to its recruitment to the
plasmamembrane and alleviating the repressive influence of the PH
domainon the kinase domain. Once at the plasma membrane, it is acted
uponby one or more upstream kinases that are themselves regulatedby
phospholipid products of PI3'-kinase (16, 17)
. A
phosphoinositide3,4,5-trisphosphate-dependent protein kinase 1 has
been shownto phosphorylate c-Akt at Thr-308, thereby increasing its
activity>30-fold (18)
. In 293 cells, rapid induction of
c-Akt activityby insulin and insulin-like growth factor-I is dependent
uponphosphorylation of two amino acids, Thr-308 and Ser-473 (16, 19). The identity of the Ser-473 kinase remains elusive.

The PI3'-kinase/Akt pathway is evolutionarily conserved andhas
been reported to play a key role in the insulin signal transduction
pathwayin Caenorhabditis elegans, where it appears to be
critical forthe inhibition of dauer arrest (20)
.
In mammalian cells, Aktis believed to be responsible for the
insulin-induced inhibitionof glycogen synthase kinase-3 and the
subsequent activationof glycogen synthase (21, 22, 23)
,
glucose uptake, and glucosetransporter-4 translocation (22, 24)
. Furthermore, Akt appearsto play a role in insulin-induced
protein synthesis by activationof eukaryotic initiation factor 2
(25)
, as well as phosphorylationand inhibition of 4E-BP1,
a repressor of mRNA translation (26)
.In addition to its
role in metabolism, Akt plays a role in theregulation of apoptosis in
metazoan organisms from Drosophila(27)
to mammals
(28, 29, 30)
. It also appears to be involvedin the growth
factor-mediated survival of neurons. Experimentswith the PC12
pheochromocytoma cell line (31)
, which is dependentupon
PI3'-kinase for its survival (32)
, and with H19-7
hippocampalneuronal cells demonstrate that pharmacological inhibitors
ofPI3'-kinase, as well as dominant-negative forms of Akt, inhibit
theactivation of Akt and induce apoptosis (33)
. The
mechanismof Akt-mediated suppression of apoptosis is unclear. However,
ithas been suggested that this may occur through Akt-induced
phosphorylationof the proapoptotic proteins BAD, caspase-9, and FKHRL1
(34,35, 36)
. Furthermore, both Fas- and Myc-induced apoptosis
(37, 38)are abrogated after activation of PI3'-kinase and
Akt (39, 40).

Of interest is the fact that Akt is a proto-oncogene
(4)
, andAkt isoforms have been shown to be overexpressed
in the MCF7breast cancer cell line (9)
. Moreover, it has
been found tobe amplified >20-fold in a gastric adenoma
(5)
, and Akt2is overexpressed in a significant
number of ovarian (41)
andpancreatic (42)
cancers. In addition, Akt appears to be a downstreamtarget for
transforming oncogenes such as Ras and v-Src(43)
.However, it is not clear how Akt participates in
oncogenesis.

To explore the ability of Akt to promote oncogenic transformation,we
have established a transformation system in Rat1 fibroblastsusing a
stably expressed, conditionally active form of Akt
(M+Akt:ER*;Ref. 44
). In brief,
M+Akt:ER* was constructed by fusing thec-Src
myristylation targeting sequence to a constitutively activeform of Akt
lacking the PH domain (M+Akt). Conditionality was
conferredin a manner similar to that described for other protein
kinases(45, 46)
by fusing M+Akt to
a modified form of the hormonebinding domain of the mouse ER (ER*)
that binds 4-HT but isrefractory to estrogen (47)
. As a
result, M+Akt:ER* is rapidlyactivated in
response to 4-HT and elicits effects that havebeen attributed to
endogenous cellular Akt (24, 44)
. In thisstudy, we
demonstrate that activation of M+Akt:ER* induces
oncogenictransformation in Rat1 fibroblasts, as manifested by
alterationsin cell morphology and the capacity to form colonies in
agarose.In addition, activation of M+Akt:ER* was
sufficient to inducecell transformation by its dual ability to
suppress apoptosisand to promote the entry and progression of cells
through thecell cycle. The latter correlated with a sustained decrease
inlevels of the cdk inhibitor p27Kip1 and an
increase in the activityof cdk2. These data are consistent with
previous reports thatPI3'-kinase and the PTEN PI3'-lipid phosphatase
participatein the regulation of cell cycle progression and apoptosis
inhuman cancer cells. Thus, we are able to directly implicateAkt in
the regulation of events ascribed previously to the PTEN/PI3'-kinaseas
a whole. We also observed that activation of
M+Akt:ER* ledto rapid activation of the p42/p44
ERK/MAP kinases; however,ERK/MAP kinase activation was not required
for the morphologicalalterations in Rat1 cells. These data indicate
that Akt hasthe capacity to regulate key biological processes that
participatein oncogenic transformation in mammalian cells and are
consistentwith a role for Akt in human tumorigenesis.

Akt Activation Induces Cellular Transformation of Rat1 Fibroblasts.
To determine the effects of Akt activation on cellular transformation,
weused retrovirus-mediated gene transfer to generate pools ofRat1
cells expressing a membrane-targeted, conditionally activeAkt/PKB
(M+Akt:ER*). As a control, we expressed a
myristylation-defectiveform of the protein
(M-Akt:ER*), which is not membrane associated
andtherefore nontransforming. The M+Akt:ER*
construct is reportedto elicit many of the downstream events
attributed to wild-typeor constitutively active forms of Akt, whereas
M-Akt:ER* doesnot (24)
. Western
blot analysis revealed that both M-Akt:ER*and
M+Akt:ER* were expressed equally well in Rat1
cells (datanot shown). Although the ability of Akt to induce changes
incell morphology in chicken fibroblasts has been reported
(48)
,here we confirm and extend these observations using
M+Akt:ER*in mammalian cells. The ability of Akt
to induce cellular transformationwas assessed in three ways: by its
ability to induce changesin cell morphology; by its ability to elicit
an altered colonymorphology; and by its ability to promote cell growth
in ananchorage-independent manner in agarose.

To determine whether the activation of
M+Akt:ER* was able toinduce changes in cell
morphology, confluent cultures of Rat1fibroblasts expressing
M+Akt:ER* (Rat1::M+Akt:ER*)
were culturedin the absence or presence of 100 nM 4-HT.
Representative datafrom one of seven experiments are shown. Within
24 h, cellscultured in the presence of 4-HT were found to be
refractileby phase-contrast microscopy. After 48 h (Fig. 1B)
, the cellsexhibited an elongated, refractile,
spindle-like morphology.In areas, these cells began to overgrow one
another, indicatingthat they had lost the ability to be contact
inhibited. In contrast,cells cultured in the absence of 4-HT (Fig. 1A)
grew to confluencein an even monolayer and displayed
normal contact inhibition.

Fig. 1. Akt activation is sufficient to induce cellular transformation of Rat1
fibroblasts. Confluent monolayers of
Rat1::M+Akt:ER* cells were split 1:3 and plated
in the absence (A) or the presence (B) of
100 nM 4-HT. M+Akt:ER*-induced morphological
transformation in Rat1 fibroblasts is dependent upon membrane
localization and 4-HT. Cells infected with either a
myristylation-defective (M-Akt:ER*; C) or a
myristylation-competent (M+Akt:ER*) form of
M+Akt:ER* (D) were cultured to confluence
and treated with 100 nM 4-HT for an additional 18 h.
E, M+Akt:ER* induced an altered colony
morphology on plastic. Cells (1 x 104) were plated in
media in the absence or the presence of 100 nM 4-HT as
indicated and cultured for 14 days, fixed with methanol, and Geimsa
stained. Conditionally active forms of Akt (M+Akt:ER*) and
Raf (EGFPRaf-1: AR), as well as a constitutively active form of Akt
(myrAkt), induced anchorage-independent growth. F,
Rat1::M+Akt:ER* fibroblasts were plated in DMEM
containing 0.35% agarose at a concentration of 1 x
103 and 1 x 104 cells/well in the absence
or presence of 500 nM 4-HT as indicated. After 14 days in
culture, cells were stained with
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and
photographed. G, similarly, Rat1 cells expressing myrAkt
or Rat1::eGFPRaf-1:AR fibroblasts were plated in agarose.
EGFPRaf-1:AR was activated by incubation with 300 nM
testosterone.

It has been suggested that the recruitment of Akt to the membraneis
essential for its activation and function (49)
. To
determinewhether membrane localization of
M+Akt:ER* is essential forits ability to induce
morphological transformation, cells expressingeither
M+Akt:ER* (Fig. 1D)
or a
myristylation-defective M-Akt:ER(Fig. 1C)
were grown to confluence and then cultured for 18h
in the presence of 100 nM 4-HT. Only cells
expressing themembrane localized M+Akt:ER*,
became morphologically altered,and lost the ability to be contact
inhibited in response to4-HT. These data indicate that membrane
localization of M+Akt:ER*is likely to be
essential for its full activation, targetingto relevant substrates and
effectors, or both.

To ascertain whether activation of M+Akt:ER*
altered the growthcharacteristics of
Rat1::M+AKT:ER* fibroblasts on
extracellularmatrix, we compared the growth of cells plated at
subconfluentdensities (1 x 104 cells/10-cm
plate) and cultured for 14 days(Fig. 1E)
. Representative
data from one of four experimentsare shown. Cells cultured in the
absence of 4-HT formed flat,diffuse colonies that displayed contact
inhibition. In contrast,cells that expressed an activated
M+Akt:ER* formed dense, multilayeredcolonies of
refractile cells. Because of their increased celldensity, these
colonies were revealed as more darkly stainingmacroscopic foci.

To determine whether the ability to elicit alterations in cellmorphology was restricted to Rat1 cells,
M+Akt:ER*expressingNIH3T3 and Rat1A
fibroblasts cell lines were derived by retrovirusinfection. Activation
of M+Akt:ER* in these cells also inducedfocus
formation at a high frequency; however, its effects onmorphology
changes in these cells were quite subtle and quiteunlike the effects
elicited by Ras or Raf (data not shown).Thus, the ability of activated
M+Akt:ER* to elicit changes incolony morphology
was not restricted to Rat1 fibroblasts.

To address whether the activation of M+Akt:ER* in
Rat1 fibroblastswas capable of inducing anchorage-independent growth,
the abilityof these cells to form colonies in agarose was examined.
Cellswere plated in agarose at 103
or
104 cells/well in the absenceor presence of 4-HT
to activate M+Akt:ER*. After 7 days in culture,
cellsexpressing M+Akt:ER* formed macroscopic
colonies in the presenceof 4-HT. After 14 days, the cultures were
stained with the vitaldye
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(Fig. 1F)
. Measured over six independent experiments, the
frequencyof colony formation induced by activated
M+Akt:ER* was 31.3± 3.8% and 0.84% ±
0.74% in the absence of 4-HT.The frequency of colony formation
induced by activated M+Akt:ER*was similar to
that induced by a constitutively active formof Akt (myrAkt). In a
separate experiment, the ability of M+Akt:ER*to
induce colony formation in Rat1 cells was compared with thatof
conditionally active EGFPRaf-1:AR and was found to be comparable
(Fig.1G)
.

Rapid, Dose-dependent Activation of M+Akt:ER* by 4-HT.
To better understand the mechanism of
M+Akt:ER* activation,the kinetics of its
induction after the addition of 4-HT weredetermined. Confluent,
serum-deprived Rat1::M+Akt:ER*
fibroblastswere treated with 300 nM 4-HT to activate
M+Akt:ER* for differentperiods of time, and its
activity was assessed by immune-complexkinase assay (Fig. 2A)
. M+Akt:ER* was activated within 15
minafter the addition of 4-HT, with maximum activation occurring
between1 and 2 h. Thereafter, the activity was sustained at peak
levelsfor the duration of the time course.

Fig. 2. Time course of M+Akt:ER* activation after treatment with
4-HT. A, Rat1::M+Akt:ER* cells
were grown to confluence, cultured in the absence of serum for 4 h, and then treated with 300 nM 4-HT for various times from
5 min to 3 h or ethanol alone (solvent control) for 3 h, at
which time cell lysates were prepared. M+Akt:ER* was
immunoprecipitated with a polyclonal anti-ER (ER) antibody. The
activity of M+Akt:ER* was measured using an immune-complex
kinase assay with histone 2B (H2B) as a substrate. The
amount of M+Akt:ER* present in each kinase reaction was
determined by Western blot analysis with the polyclonal anti-ER
antibody. B, M+Akt:ER* activation is
dependent upon the concentration of 4-HT.
Rat1::M+Akt:ER* cells were cultured as described
above and then stimulated with various concentrations of 4-HT from 3 to
1000 nM or with 20% FCS as indicated. An Akt
immune-complex kinase assay was performed as described above with H2B
as substrate (lower panel). The phosphorylation status
of serine residue 473 was determined by Western blot analysis with an
anti-phosphoserine 473 antibody (middle panel). The blot
was subsequently stripped, and Western blot analysis with the
polyclonal anti-ER antibody was used to determine the amount of
M+Akt:ER* in each lane.

To determine effective doses for the stimulation of
M+Akt:ER*kinase activity, confluent,
serum-deprived Rat1::M+Akt:ER*
fibroblastswere stimulated with various concentrations of 4-HT from 0
to1000 nM for 1 h. M+Akt:ER*
activity increased with the concentrationof 4-HT added and was maximal
between 30 and 100 nM 4-HT (Fig.2B)
. Increases
in the dose of 4-HT beyond 100 nM did not
significantlyincrease the kinase activity of
M+Akt:ER*. The results indicatethat the kinase
activity of M+Akt:ER* may be titrated by altering
theconcentration of 4-HT added to cell culture media.

It has been reported that Akt must be phosphorylated on boththreonine
residue 308 and serine residue 473 to be fully activated(16, 19)
. M+Akt:ER* became phosphorylated on
Ser-473 uponstimulation with 4-HT. This phosphorylation was detectable
at4-HT concentrations of 100-1000 nM. In addition,
treatment ofRat1::M+Akt:ER* cells with
a high concentration of serum wasalso able to induce
M+Akt:ER* phosphorylation and activity.These
data are consistent with the notion that
M+Akt:ER*, similarto wild-type Akt, may still in
part be regulated by PI3'-lipids.However, the induction by serum was
not as robust as with 4-HT.When given in combination, serum and 4-HT
treatment led to hyperactivationof M+Akt:ER*
kinase activity (data not shown). Taken together,these results
indicate that M+Akt:ER* activity is rapidly
inducedin response to 4-HT and is titratable to levels higher than
observedafter acute stimulation with serum.

An apparent discrepancy in this experiment is the activationof
M+Akt:ER*, as measured by kinase assays in the
absence ofdetectable Ser-473 phosphorylation at 30 and 100
nM 4-HT. Itis not clear whether this reflects an
uncoupling of Ser-473phosphorylation from
M+Akt:ER* activation or a reflection ofthe
sensitivity of the phospho-Ser-473-specific antiserum. Themost likely
explanation for the apparent discrepancy is thatthe Akt immune-complex
kinase assay may simply be more sensitivethan the phosphospecific
antibody. At lower levels of Akt activitythere may be a
correspondingly low level of Ser-473 phosphorylationthat is not
detectable with the phospho-specific antibody.

Activation of M+Akt:ER* Protects Rat1 Cells from
Apoptosis.
Akt is believed to transduce survival signals from a numberof
cytokine receptors as well as survival signals elicited bythe binding
of integrins to the extracellular matrix (28)
.Thus, the
prevention of apoptosis may play an important rolein promoting the
anchorage-independent growth of
Rat1::M+Akt:ER*cells in agarose and
thereby contribute to M+Akt:ER*-inducedcellular
transformation. Accordingly, the effect of
M+Akt:ER*activation on apoptosis was determined
after the removal ofboth serum and extracellular matrix survival
signals. Apoptosiswas measured by Annexin V-FITC and PI staining, and
flow cytometrywas used to quantitate the percentage of apoptotic cells
inthe culture. Representative data from one of four experimentsare
shown (Fig. 3)
. Attached Rat1::M+Akt:ER* fibroblasts
culturedin the absence of serum for 18 h either with or without
activatedM+Akt:ER* were alive and nonapoptotic
because they excludedPI and were negative for Annexin V staining (Fig. 3, left panel
).These results indicated that Rat1 cells are
protected from apoptosis,even in the absence of serum, as long as they
remain attachedto the cell culture dish. As a result, the activation
of M+Akt:ER*had little or no effect on cell
viability under these conditions.However, when cells were detached
from the monolayer and culturedin suspension in the absence of serum,
activation of M+Akt:ER*had a significant effect
on cell viability (Fig. 3, right panel
).Cells cultured in
suspension in which M+Akt:ER* was inactive
underwentprogrammed cell death with 42% of cells becoming Annexin
V positivein 12 h. In contrast, cells in which
M+Akt:ER* was activatedwere, at least in part,
rescued from apoptosis because only25% of these cells were Annexin V
positive. Therefore, in Rat1fibroblasts, activation of
M+Akt:ER* was able to inhibit apoptosisinduced
by removal of cells from extracellular matrix. A timecourse for the
onset of apoptosis under these conditions wasdetermined after the
placement of these cells in suspensionin the absence or presence of an
activated M+Akt:ER*. Asynchronouslygrowing cells
were cultured in suspension in the absence ofserum for 0, 6, 12, 18,
and 24 h with or without 4-HT to activate
M+Akt:ER*(Fig. 3E)
. PI staining and
flow cytometry analysis of fragmentednuclei were used to determine the
percentage of apoptotic cellsin culture at the indicated times. Our
results indicate thatafter 24 h, the percentage of apoptotic
cells increased from10% to almost 40%. However, in the presence
of activated M+Akt:ER*,by 24 h the
percentage of apoptotic cells was 17%. Therefore,in Rat1
fibroblasts, activation of M+Akt:ER* was able to
delaythe onset of apoptosis. These observations are consistent with
theability of M+Akt:ER* to promote
anchorage-independent growth(Fig. 1F)
and suggest that the
ability of activated M+Akt:ER*to induce Rat1
cells to grow in agarose may, at least in part,be dependent on its
ability to prevent apoptosis in these cells.To elucidate the molecular
mechanisms underlying the preventionof apoptosis in
Rat1::M+Akt:ER* fibroblasts, the
expressionof candidate cell survival and proapoptotic gene products
wasexamined by Western blot analysis after activation of
M+Akt:ER*.There was no change in the expression
of the antiapoptotic proteinsBcl-2 or Bcl-xL.
Thus, our findings here confirm previous worksuggesting that Akt does
not alter Bcl-2 or Bcl-xL expression
(50)
.In addition, we found that there was no change in
the expressionof the proapoptotic protein Bax. It remains to be seen
whetherM+Akt:ER* phosphorylates to inactivate
the proapoptotic proteinsBad, caspase-9, or FKHRL1 in these cells,
as has been reportedpreviously in other cells (34, 35, 36)
.

Fig. 3. M+Akt:ER* protects Rat1 cells from apoptosis. Left
panel, Rat1::M+Akt:ER* cells were grown
to confluence, left untreated (A) or treated with 100
nM 4-HT (C), and cultured for 18 h in
the absence of serum, at which time they were detached and stained with
Annexin V-FITC and PI. Right panel, confluent monolayers
were trypsinized, left untreated (B) or treated with 100
nM 4-HT (D), and cultured in suspension in
the absence of serum for 12 h. Flow cytometry was used to
determine Annexin V and PI staining and to determine whether cells were
undergoing apoptosis. Live cells are found in the lower left
(LL) quadrant, cells in the early stages of apoptosis
are found in the lower right (LR) quadrant, and cells
late in the apoptotic program are found in the upper right
(UR) quadrant. E, asynchronously growing
Rat1::M+Akt:ER* cells were placed in suspension
on ultra-low attachment plates in the absence of serum. PI staining and
flow cytometry were used to score the percentage of apoptotic cells
with fragmented nuclei both in the absence (-4-HT) or
presence (+4-HT) of M+Akt:ER* activation.
The time indicated represents the number of hours the cells were in
suspension.

M+Akt:ER* Activation Is Sufficient to Induce DNA
Synthesis and Cell Cycle Progression.
Increased cell cycle progression may also contribute to the
growthof transformed Rat1::M+Akt:ER*
fibroblasts. To determine theproliferative potential of these cells,
clonal populations wereestablished by selecting individual colonies
growing in agarose.These SAR1 cells displayed rapid changes in cell
morphology,becoming transformed within 24 h after activation of
M+Akt:ER*(data not shown). The high frequency
(>30%) with which theSAR1 cells form colonies in soft agar would
suggest that thesecells are representative of the population at large
and mayarise as a result of the levels of
M+Akt:ER* expression ratherthan through the
acquisition of additional transforming events.To elucidate the role,
if any, of activated M+Akt:ER* in cellcycle
progression and to determine whether activation of
M+Akt:ER*in quiescent SAR1 fibroblasts was
sufficient to induce DNA synthesis,SAR1 fibroblasts rendered quiescent
by culture in the absenceof serum were treated with 100 nM
4-HT to activate M+Akt:ER*.Induction of DNA
synthesis was assessed by the incorporationof BrdUrd into DNA, which
was detected by antibody stainingand flow cytometry. Representative
data from one of eight separateexperiments are shown (Fig. 4, A and B)
. In the absence of
M+Akt:ER*activation, 16% of SAR1 cells had
incorporated BrdUrd (Fig.4A)
. In contrast, after the
activation of M+Akt:ER* for 18h, 44% of
the cells were BrdUrd positive (Fig. 4B)
. These data
indicatethat activation of M+Akt:ER* is
sufficient to induce DNA synthesisin Rat1 cells, even in the absence
of serum.

Fig. 4. Akt activation is sufficient to induce cell cycle progression in Rat1
cells. SAR1 cells were grown to confluence and rendered quiescent by
culture in the absence of serum for 5 days. The cells were then treated
with ethanol (A) or with 4-HT (B) for an
additional 18 h. The induction of DNA synthesis was assessed by
BrdUrd incorporation after activation of M+Akt:ER*. Region
3 (R3) is representative of cells that have incorporated
BrdUrd as they progressed through S phase. Cell cycle analysis after
activation of M+Akt:ER* is shown. Confluent monolayers of
SAR1 cells were rendered quiescent (as above) and then treated with
ethanol (C) or with 4-HT (D) for 18 h. The cells were stained with PI, and cell cycle analysis was
performed. M1, apoptotic cells; M2,
G0-G1 phase cells; M3, S-phase
cells; M4, cells in the G2-M phase of the
cell cycle.

To confirm the data obtained by BrdUrd labeling, SAR1 cellsas well as
the pooled population of
Rat1::M+Akt:ER* cells wererendered
quiescent and M+Akt:ER* activated. The cells were
stainedwith PI, and cell cycle analysis was performed using flow
cytometry.Representative data of five separate experiments for the
SAR1population are shown (Fig. 4, C and D)
.
Prior to activationof M+Akt:ER* (Fig. 4C)
, 83% of the cells were in
G0-G1 phase(M2), 11% of
the cells were in S phase (M3), and 5% of the cellswere in
G2-M phase (M4). In marked contrast, after
activationof M+Akt:ER* (Fig. 4D)
,
48% of the cells were in
G0-G1 (M2),30% of the
cells were in S phase (M3), and 19% of the cellswere in
G2-M phase (M4). Similar results were obtained
fromthe pooled population (data not shown). These data confirm the
resultsof the BrdUrd experiment that activation of
M+Akt:ER* is sufficientto induce the progression
of serum-deprived Rat1 cells throughthe cell cycle.

M+Akt:ER* Activation Induces Cyclin E/cdk2 Kinase
Activity and Degrades p27Kip1.
The cell cycle is regulated through the periodic synthesis and
destructionof cyclins that associate with and regulate the activity of
cdks.The activity of the cyclin E/cdk2 complex is required for the
G1to S-phase transition (51, 52)
.
The activity of this complexis regulated both by the levels of
expression of cyclin E proteinas well as by the Cip/Kip family of cdk
inhibitors, which binddirectly to and inhibit cyclin E/cdk2
(53)
. Expression of p27Kip1is
growth inhibitory, and genetic evidence suggests that micelacking
p27Kip1 are abnormally large, have multiple organ
hyperplasia,and are predisposed to pituitary tumors
(54, 55, 56)
. Therefore,to address the molecular mechanism by
which activation of M+Akt:ER*promoted the entry
of cells into S phase, SAR1 cells as wellas the pooled population of
Rat1::M+Akt:ER* cells were usedto
determine the levels of key regulators of the
G1-S-phasetransition. Both the pooled population
and SAR1 cells gave similarresults. Representative data for cyclin E
expression, the kinaseactivity of the cyclin E/cdk2 complex, as well
as the levelof p27Kip1 expression in SAR1 cells
after activation of M+Akt:ER*are shown (Fig. 5)
. Consistent with the entry of cells intoDNA synthesis in Fig. 4
,
cyclin E/cdk2 kinase activity wasfound to be increased after
activation of M+Akt:ER*. However,the levels of
cyclin E in complex with cdk2 were unchanged,indicating that increased
cyclin E expression was not responsiblefor the increased cdk2 kinase
activity. In contrast, the levelof p27Kip1 was
found to be significantly decreased after theactivation of
M+Akt:ER*. Our findings are consistent with
previouswork that suggests a role for the PTEN/PI3'-kinase pathway in
regulatingp27Kip1 expression (57)
.
Furthermore, our results directlysuggest that the effects of the
PTEN/PI3'-kinase pathway onp27Kip1 may be
mediated through Akt. Because Rat1 cells do notexpress
p21Cip1(58)
, the loss of
p27Kip1 from the cyclin E/cdk2complex likely
plays an important role in the M+Akt:ER*-induced
entryof cells into S phase. Consequently, the repression of
p27Kip1expression may be a contributing factor
to the oncogenic transformationof cells by Akt.

Fig. 5. Activation of M+Akt:ER* induces cdk2 kinase activity and
loss of p27Kip1 from the cdk2 complex. Quiescent SAR1 cells
were treated with 4-HT to activate M+Akt:ER* for the number
of hours indicated, at which point the cells were lysed and cdk2 kinase
activity was determined using an immune-complex kinase assay with
histone H1 (H1) as a substrate. As a control, cells were
treated with ethanol and cultured for 24 h (24-)
. The
levels of cyclin E, cdk2 in the immunoprecipitates, and the expression
of p27Kip1 in cell lysates were determined by Western
blot.

M+Akt:ER* Activates the ERK/MAP Kinase Pathway.
Because the Raf-MEK-ERK/MAP kinase pathway has been shown tobe
oncogenic in Rat1 cells, we wanted to determine whether thep42/p44
ERK/MAP kinases were activated in response to
M+Akt:ER*activation. Confluent cultures of
quiescent SAR1 fibroblastswere treated with 100 nM 4-HT to
activate M+Akt:ER* for variouslengths of time
from 5 min to 3 h, and the activity of ERK1and ERK2 was
determined by Western blot using a phospho-specificanti-active ERK
antibody. In response to M+Akt:ER* activation,
ERK1and ERK2 were activated between 30 min and 1 h, with maximum
activationin this experiment occurring after 3 h (Fig. 6A)
. In parentalRat1 cells, 4-HT alone did not induce the
ERK/MAP kinase pathway(data not shown). The rapid time course of
ERK/MAP kinase activationafter activation of
M+Akt:ER* suggests a direct stimulationof this
pathway but does not rule out other possibilities, suchas the rapid
release of autocrine growth factors.

Fig. 6. M+Akt:ER* activates ERK/MAP kinase in a
MEKdependent manner. A, confluent monolayers of
SAR1 cells were cultured in the absence of serum for 4 h and were
subsequently treated with 4-HT for various times from 5 min to 3 h
as indicated. ERK/MAP kinase activity was determined by Western blot
analysis with a monoclonal anti-active-phospho-ERK/MAP kinase antibody.
B, to determine whether activation of ERK/MAP kinase by
M+Akt:ER* was dependent on MEK activity, an immune-complex
kinase assay for ERK/MAP kinase activity was performed. SAR1 cells were
cultured as above and treated with 4-HT (Akt) or PDGF (10 ng/ml) in the
absence (DMSO as solvent control) or the presence of the MEK inhibitor
PD098059. The p44 isoform of ERK/MAP kinase was immunoprecipitated with
a polyclonal anti-p44 ERK/MAP kinase antibody. The kinase assay was
subsequently Western blotted with a polyclonal anti-p44 ERK/MAP kinase
antibody to determine the amount of p44 ERK/MAP kinase in each
reaction. C, confluent (Con.) and
subconfluent (Sub.) monolayers of parental Rat1 cells
and Rat1 cells expressing constitutively active Akt
(myrAkt) were harvested, and the activity of the p42/p44
ERK/MAP kinases was measured using an immune-complex kinase assay using
myelin basic protein as a substrate. As a control, SAR1 cells that were
either untreated or treated with 4-HT (100 nM) were
harvested. The kinase reactions were subsequently reprobed
with antisera that recognize p42/p44 ERK/MAP kinase to determine
equal loading of the reactions. IP, immunoprecipitation;
WB, Western blot.

To determine whether the M+Akt:ER*-induced
activation of theERK/MAP kinase pathway was dependent on MEK,
confluent monolayersof quiescent SAR1 fibroblasts were treated with a
pharmacologicalinhibitor of MEK PD098059 for 15 min. The cells were
then stimulatedwith PDGF or with 4-HT. PDGF is known to activate the
ERK/MAPkinases in a MEK-dependent manner and therefore serves as an
appropriatepositive control. Both PDGF and activated
M+Akt:ER* were ableto rapidly activate the
ERK/MAP kinases in these cells (Fig.6B)
. In both cases, the
activation of the ERKs was inhibitedby PD098059. Therefore, we
conclude that the activation of ERK/MAPkinase activity by
M+Akt:ER* is dependent upon the activityof MEK.

To determine whether the observed activation of ERK/MAP kinasewas a
direct result of M+Akt:ER* activation or an
unusual propertyof the Akt:ER* fusion protein, Rat1 cells expressing a
constitutivelyactive form of Akt (myrAkt) were generated. Confluent as
wellas subconfluent cultures of these cells expressing myrAkt andSAR1
cells cultured with 4-HT were found to have elevated levelsof ERK/MAP
kinase activity (Fig. 6C)
. Consequently, these dataindicate
that activation of p42/p44 ERK/MAP kinases in Rat1cells is a property
of constitutive and conditionally activeforms of Akt.

Morphological Transformation by M+Akt:ER* Is Not MEK
Dependent.
To determine whether the effects of Akt on cell morphology were
mediatedthrough the ERK/MAP kinase pathway, we generated SAR1 cells
thatalso express EGFPRaf-1:AR, a conditionally active form of Raf
thatis regulated by androgens and their analogues. In these cells,we
have the capacity to activate M+Akt:ER using
4-HT, EGFPRaf-1:ARusing testosterone, and both proteins by the
co-addition of4-HT and testosterone. Activation of either Raf or Akt
elicitedmorphological alterations in these cells (Fig. 7, AC)
.Pre-addition of PD098059 to inhibit MEK
significantly inhibitedthe effects of Raf activation on Rat1 cell
morphology (Fig.7
, compare B and E). However, in
this experiment, PD098059had little or no effect on the ability of Akt
to elicit morphologicalalterations in these cells (fig. 7
, compare
C and F). Examinationof data from nine separate
experiments indicated that occasionallya modest effect of PD098059 on
Akt-induced morphological effectswas observed but always much less
than the effects of the compoundon Raf-transformed cells. These data
suggest that the abilityof Akt to activate the ERK/MAP kinase pathway
is likely dispensablefor morphological transformation. As yet we have
not addressedwhether the ERK/MAP kinase pathway participates in the
effectsof Akt on apoptosis or cell cycle progression.

Fig. 7. Morphological transformation of cells by M+Akt:ER* does not
require MEK activity. Confluent cultures of SAR1 fibroblasts infected
with eGFPRaf: AR were split 1:3 and treated with DMSO or PD098059
(PD) for 15 min. Subsequently, 300 nM
testosterone to activate EGFPRaf:AR or 100 nM 4-HT to
activate M+Akt:ER*, or ethanol as solvent control, were
added to cells, and the cells were cultured for an additional 18 h.

Considerable evidence indicates that PI3'-kinase regulated
signalingpathways play an important role in a variety of cellular
processes(59, 60, 61)
and that the protein kinase Akt is an
importantmediator of these effects (21, 22, 23, 24, 25, 26)
. For example,
inductionof cyclin D1 by growth factors and oncogenes in NIH 3T3 cells
isPI3'-kinase dependent, thus suggesting that the PI3'-kinase/Akt
pathwaymay play an important role in the control of cell cycle
progression(62, 63)
. Moreover the role of PI3'-kinase in
the suppressionof apoptosis and the ability of Akt to directly
phosphorylatekey mediators of the apoptotic response provide strong
circumstantialevidence for the importance of the PI3'-kinase-Akt
pathway inthe aberrant behavior of cancer cells.

Direct evidence for an important role of PI3-kinase and Aktin
oncogenic transformation may be inferred from the observationthat
activated forms of these proteins have been isolated fromacutely
transforming retroviruses of mice and chickens. An avianretrovirus
encoding p3K, an activated form of the catalyticsubunit of
PI3'-kinase, causes hemangiosarcomas in chickensand transforms chick
embryo fibroblasts in culture (64)
. Amurine retrovirus
encoding v-Akt causes T-cell lymphomas (7)and accelerates
the development of leukemias in severe combinedimmunodeficient mice
(65)
, and overexpression of Akt2 in murinefibroblasts
elicits cellular transformation (66)
. Furthermore,a
mutant form of the p85 regulatory subunit of PI3'-kinase
(p65-PI3'-kinase;Ref. 67
), as well as a conditionally
active form of p110 (p110:ER;Ref. 68
), promotes cellular
transformation in mammalian cellsin culture.

A role for the PI3'-lipids in human cancer is most stronglyimplied by
the fact that PTEN, a lipid phosphatase that selectively
dephosphorylatesthe 3'-position of the inositol headgroup, is a
tumor suppressorgene that is deleted or mutated in both sporadic
cancer andin inherited cancer predisposition syndromes. For example,
glioblastomaslacking PTEN display elevated PI3'-lipid
production and a correspondinglyhigh level of Akt activity
(69)
. In addition, a number of ovariancancers display
amplification of the gene encoding the p110-catalyticsubunit of
PI3'-kinase (70)
. Various Akt isoforms have beenreported
to be amplified or overexpressed in breast cancer cells
(9)
,gastric adenomas (5)
, ovarian
(41)
, and pancreatic (42)
cancers.Finally
the reported ability of oncogenic Ras to activate PI3-kinasein
vitro(71)
may provide another link between this
signalingpathway and alterations detected in human cancer.

To further study the effects of Akt in mammalian cells, we
haveestablished a conditional transformation system for this oncogene
usingM+Akt:ER, which requires the estrogen
analogue 4-HT for itsactivity. Our results suggest that Akt may
activate a numberof pathways leading to oncogenic transformation of
Rat1 cells.Activation of Akt in Rat1 cells elicited significant
alterationsin cell morphology, loss of contact inhibition, promoted
anchorage-independentproliferation in agarose, protected cells from
apoptosis, andpromoted the reentry of serum-deprived cells into the
cell divisioncycle.

Progression of cells into S phase requires the activity of cyclin
E/cdk2complexes. The ability of Akt to promote cell cycle progression
correlatedwith its ability to repress the expression of
p27Kip1, an inhibitorof the cyclin E/cdk2
complex (72, 73)
. Proliferating cells(74)
as
well as many types of human cancers have reduced expressionof
p27Kip1(75, 76, 77, 78)
. In addition, there
is mountingevidence to suggest that the progression and prognosis of
manyforms of cancer are inversely correlated to the levels of
p27Kip1(79, 80, 81, 82)
. Expression of
p27Kip1 in MCF7 cells reducedtheir capacity for
anchorage-independent cell growth and theirtumorigenic capacity in
xenografts (83)
. However, the mechanismsthat influence
p27Kip1 expression in tumors have not been
extensivelyexplored. Indirect evidence suggests the involvement of the
PTEN-PI3'-kinase-Aktpathway in certain circumstances. Ectopic
expression of PTENin glioblastoma cells that lack PTEN expression led
to decreasedAkt activity and increased p27Kip1
expression and localizationin cyclin E/cdk2 complexes
(84)
. Furthermore Pten-/-
embryonicstem cells have increased PI3'-lipid production, a high level
ofAkt activity, and decreased p27Kip1 expression
(85)
. However,it has not been demonstrated previously
that Akt can directlyinfluence p27Kip1
expression. At present, it is not clear whetherdecreased
p27Kip1 expression is a consequence of a direct
effectof Akt on p27Kip1 or whether it may be a
consequence of inducedcyclin A synthesis. The ubiquitination and
subsequent destructionof p27Kip1 is reported to
be initiated by cdk2-mediated phosphorylationof
p27Kip1 on Thr-187 (74, 86)
. If Akt
activation leads toelevated cyclin A expression, it might provoke
sufficient cdk2activation to promote p27Kip1
destruction. Alternatively, Aktmay activate a signaling pathway that
directly represses p27Kip1expression. Analysis
of the effects of Akt on p27Kip1 mRNA andprotein
expression as well as the use of various p27Kip1
mutantswill allow this question to be addressed.

Perhaps surprisingly, we observed that both constitutive and
conditionallyactive forms of Akt activated the ERK/MAP kinase pathway
inRat1 cells. Although the mechanism of activation remains unclear,
thereis clear precedent from other studies for a role for PI3'-kinase
inERK/MAP kinase activation (87, 88, 89, 90, 91, 92, 93)
. Forexample,
PDGF-induced ERK/MAP kinase activation is inhibitedby the PI3'-kinase
inhibitors wortmannin and LY294002 underconditions where PDGF is
limiting (88)
. Furthermore, activationof the Raf-MEK-ERK
pathway after integrin engagement is alsoinhibited by PI3'-kinase
inhibitors as well as by a dominant-negativeform of p85
(94)
. In this case, the PI3'-kinase sensitive stepis in
the activation of Raf-1. Recent evidence suggests Aktmay directly
phosphorylate Raf-1, leading to its inactivationduring differentiation
(95, 96)
, which stands in contrastto the observations
presented here. There is compelling evidenceto suggest that both the
PI3'-kinase/Akt pathway as well asthe Raf-MEK-ERK pathway are both
required for the expressionof key cell cycle regulators (63, 97)
. Our data are consistentwith these data and further suggest
that the cell type-specificinterplay or cross-talk between these
oncogenic pathways mayplay an important role in cell proliferation,
differentiation,and transformation. Our future studies will address
whetherAkt directly phosphorylates Raf-1 in Rat1 cells and determine
theeffects of Akt on other components of the Ras-activated Raf-MEK-ERK
pathway.Despite the ability of Akt to activate the ERK/MAP kinase
pathway,it is clear that other Akt-activated pathways must contribute
tothe effects observed in Rat1 cells because the MEK inhibitor
PD098059had only a modest effect on Akt-induced alterations in cell
morphology.To address this, we are currently examining the
phosphorylationof other putative Akt targets in Rat1 cells.

The utility of steroid hormone-regulated protein kinases tostudy
intracellular signaling pathways is now well established.In addition,
the ability to use steroid hormone binding domainsfor androgens,
mineralocorticoids, progesterone, and othersallows cell lines to be
constructed in which different signalingpathways are under the control
of different steroid hormones.To this end, we have constructed cells
in which Raf or Akt maybe activated selectively using testosterone or
4-HT, respectively,or both pathways may be coordinately activated by
the additionof both hormones. Such cells will permit the analysis of
theeffects of signal pathway activation either alone or in conjunction
withother signaling pathways. By this means, it will be possibleto
investigate gene regulation and downstream biological effectsthat
occur because of cooperative activation of multiple signalingpathways.

Construction of Retrovirus Expression Vectors.
The construction of retrovirus vectors for the expression of
M+Akt:ER*in mammalian cells has been described
previously (44)
. To constructan androgen-regulated form
of Raf-1, DNA sequences encodingthe hbAR, supplied by Dr. Hartmut
Land (University of RochesterSchool of Medicine and Dentistry,
Rochester, NY; Ref. 98
),were subjected to 10 cycles of
PCR to introduce an EcoRI siteat the 5' end and a
ClaI site at the 3' end of the coding sequence.The
resulting PCR fragment was subcloned into pCRII (Invitrogen).Because
of the presence of an internal EcoRI site within the
sequencesencoding hbAR, this plasmid was subjected first to partial
digestionwith EcoRI, followed by digestion to completion
with ClaI togenerate a 0.85-kb fragment encoding the entire
hbAR region.Retrovirus vectors (pBabepuro3 and pWZLblast3) encoding
EGFPRaf-1:ER(46, 99)
were digested with
EcoRI and ClaI to remove sequencesencoding the
hormone binding domain of the estrogen receptor.Ligation of the
EcoRI-ClaI fragment of hbAR led to the generation
ofretrovirus vectors encoding EGFPRaf-1:AR. When EGFPRaf-1:AR
wasexpressed in cells, the activity of the MEK/MAP kinase pathwayis
regulated by the addition or subtraction of testosteroneor the
synthetic androgen analogue R1881 (DuPont NEN) to thecell culture
media (98)
. Further details of these constructionsare
available on request.

Soft agar-selected
Rat1::M+Akt:ER* colonies were used to
derivethe SAR1 clonal cell line. To generate these lines, cells from
thepooled population were plated and cultured in DMEM containing
0.35%(w/v) agarose in the presence of 4-HT until colonies were
visible.Individual colonies were selected at random, the agarose was
removedby pipetting with media, and the colony was plated onto the
wellof a 24-well dish for subsequent expansion.

SAR1 cells were superinfected with a virus encoding
EGFPRaf-1:ARto produce the SAR1-Raf cells, which express the
4-HT-responsiveM+Akt:ER* and androgen-responsive
EGFPRaf-1:AR. These cellswere cloned as single cells using a
fluorescence-activated cellsorter (Becton Dickinson).

DNA Synthesis and Apoptosis Assays.
DNA synthesis was assessed by the incorporation of BrdUrd intocellular
DNA as described previously (99, 101)
. Briefly, cellswere
rendered quiescent by culture in DMEM + BSA/linoleic acidfor 5 days
with media changed every 24 h. After quiescence,the cells were
treated as described in the text. BrdUrd wasadded to the cells to a
final concentration of 50 µM,and the incubation
continued for an additional 18 h. Ethanol-fixedcells were stained
with an anti-BrdUrd antibody (Becton Dickinson).After staining, the
cells were washed and analyzed using a BectonDickinson FACScan.

Apoptosis was assessed after activation of
M+Akt:ER* by stainingwith Annexin V-coupled to
FITC, as detailed in the ApoAlertApoptosis kit protocol (Clontech
Laboratories). To determinewhether cells were undergoing
apoptosis, flow cytometry wasused to determine Annexin V and PI
staining. Live cells arefound in the lower left quadrant, cells in the
early stagesof apoptosis are found in the lower right quadrant, and
cellslate in the apoptotic process are found in the upper right
quadrant(102, 103)
. In addition, PI staining for cell
cycle analysisand scoring of the
sub-G0-G1 population was
also used.

We thank Drs. Douglas Woods, Emma Lees, David Parry, David
Stokoe,and Harmut Land, as well as the members of the McMahon lab,for
the provision of reagents and for useful comments and discussionsover
the course of this work.

Footnotes

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